The normal human diploid fibroblast-like finite cell line WI-38 and the SV40-transformed continuous subline WI-38-VA13A can now be grown in serum-free hormone-supplemented media. These media are composed of MCDB-104 and platelet derived growth factor (PDGF) (3 micrograms/ml), epidermal growth factor (EGF) (100 ng/ml), insulin (INS) (5 micrograms/ml), transferrin (TRS) (5 micrograms/ml) and dexamethasone (DEX) (55 ng/ml), and MCDB-104 and PDGF, INS, TRS, DEX and fibronectin (FN) (3 micrograms/ml) respectively. Cells grown in these serum-free media and in serum supplemented media have essentially the same growth characteristics. In the case of normal WI-38 cells, EGF acts as a principal mitogen during the first 6 hours of GO/G1 to initiate DNA synthesis 6 or 7 hours later. PDGF has little or no effect at low cell densities, however, as cells approach confluency PDGRF plays some role in overcoming density inhibition of growth. WI-38-VA13A cells lose their growth requirment for exogenously supplied EGF. They acquire, however, a dependence on PDGF and FN. These cell lines serve as models of senescence and carcinogenesis respectively. The experiments described in this proposal utilize these defined media to study cell proliferation in normal, senescent and transformed human cells. We will determine the optimum concentrations of each of the hormones and growth factors as a function of the cell's proliferative potential, and examine the regulation of DEX and EGF receptors throughout the cell cycle. The effect of each hormone and growth factor on cell cycle phase progression will also be studied. Finally, these systems will be used to characterize and isolate a normal cell-produced growth factor(s) whose synthesis and/or secretion is known to be under DEX control, and similarly to study the nature of conditioning factor(s) produced by transformed cells. Thus, we propose to study the regulation of cell proliferation in well-characterized populations of normal, senescent and transformed human cells.